Since its introduction in 1999, West Nile virus (WNV) has become the leading cause of arboviral encephalitis in the United States. Although a number of vaccine candidates are currently being evaluated, the development of a safe and effective vaccine against WNVNY99 remains a high priority. The Australian virus Kunjin (WNVKUN) is closely related to the WNVNY99 strain but is only rarely been associated with a clinical disease in man, despite a high frequency of infection in endemic areas. Immunization of mice and American crows with WNVKUN was shown to provide complete protection against subsequent challenge with a lethal dose of WNVNY9g. The largely asymptomatic infection in humans, and the efficacy of vaccination in mice and birds, makes WNVKUN an attractive vaccine candidate against WNVNY99. However, further attenuation of WNVKUN or generation of an attenuated version of the WNVNY9g strain is desirable for increasing the safety profile of these vaccines. We have recently identified several proteins in WNVKUN as key players in the virus's strategy to evade innate immune response. This application is aimed at the rational development of the attenuated WNV vaccine by further mapping mutations in viral proteins that disable WNV virus's ability to suppress IFN-?/[unreadable] response and determining the magnitude of the attenuation and the induction of protective immune responses delivered by one or more of such mutations. The following specific aims have been designed to achieve this goal: (1) To generate WNV mutants that induce increased levels of IFN-a/p by disabling the virus's ability to suppress IFN-[unreadable] transcription. Mutations shown to result in disabling activities of individually expressed viral proteins in inhibition of IFN-?/[unreadable] production will be analysed for their effect on induction of IFN-?/[unreadable] by the mutant WNV viruses, and the viruses containing the best combinations of mutations will be selected for further testing in animal models in Aim 3. (2) To generate WNV viruses with increased sensitivity to IFN-?/[unreadable] by disabling virus's ability to inhibit IFN-o/p signaling. WNV viruses with increased sensitivity to IFN-?/[unreadable] will be generated in the presence of a chemical mutagen and selected for decreased replication in the presence of IFN-?/[unreadable]. The mutations coffering IFN-?/[unreadable] sensitivity in selected viruses and/or in individually expressed viral proteins will be determined and incorporated into the WNV infectious clones for further testing in animals in Aim 3. (3) Evaluate the attenuation and immunogenecity of WNV viruses and infectious WNV plasmid DNAs containing mutations allowing increased production of and increased sensitivity to IFN-?/[unreadable]. Mutant viruses and infectious DNAs will be evaluated for attenuation and induction of protective immune responses against WNVNY99 challenge in mice and American crows and for transmission efficiency by a mosquito vector. The approach and findings obtained in this project should also be applicable to the design of attenuated vaccines against other flaviviruses from the NIAD Category A, B, and C pathogens, e.g., Dengue, Japanese encephalitis, and tick-borne encephalitis viruses.